Species specific method for the PCR detection of phythophthora

Phythophthora species which infect potatoes may result in the devastating disease potato late blight or in pink rot. Primers specific for Phythophthora infestans (late blight), and for Phytophthora erythroseptica and Phytophthora nicotianae (pink rot) have been designed which are useful for detecting the presence of the microorganisms by polymerase chain reaction methods. The primers were derived from the internal transcribed spacer region of Phytophthora ribosomal DNA and may be used to confirm the presence of the microorganisms or to distinguish among them.

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Claims

1. An oligonucleotide primer having the sequence 5'-CGATTCAAATGCCAAGCTAAAG-3' (SEQ ID NO: 1).

2. A primer set comprising oligonucleotides having the sequences 5'-CGATTCAAATGCCAAGCTAAAG-3' (SEQ ID NO: 1) and 5'-GCATCGATGAAGAACGCAGC-3' (SEQ ID NO: 2).

3. An oligonucleotide primer having the sequence 5'-CTGTTCCGGCGTAAGCTGG-3' (SEQ ID NO: 3).

4. A primer set comprising oligonucleotides having the sequences 5'-CTGTTCCGGCGTAAGCTGG-3' (SEQ ID NO: 3) and 5'-TCCTCCGCTTATTGATATGC-3' (SEQ ID NO 4).

5. An oligonucleotide primer having the sequence 5'-ATTCAAAAGCCAAGCCACCG-3' (SEQ ID NO: 5).

6. A primer set comprising oligonucleotides having the sequences 5'-ATTCAAAAGCCAAGCCACCG-3' (SEQ ID NO: 5) and 5'-GCATCGATGAAGAACGCAGC-3' (SEQ ID NO: 2).

7. A method of detecting Phytophthora species by polymerase chain reaction, said method comprising

a) providing a test sample suspected of containing said Phytophthora or the DNA of said Phytophthora,
b) amplifying the DNA of said Phythophthora with a primer set comprising oligonucleotides having the sequences 5'-CGATTCAAATGCCAAGCTAAAG-3' (SEQ ID NO: 1) and 5'-GCATCGATGAAGAACGCAGC-3' (SEQ ID NO: 2), oligonucleotides having the sequences 5'-CTGTTCCGGCGTAAGCTGG-3' (SEQ ID NO: 3) and 5'-TCCTCCGCTTATTGATATGC-3' (SEQ ID NO: 4) or oligonucleotides having the sequences 5'-ATTCAAAAGCCAAGCCACCCCG-3' (SEQ ID NO: 5) AND 5'-GCATCGATGAAGAACGCAGC-3' (SEQ ID NO: 2), and
c) detecting the presence of amplified DNA as an indication of the presence of Phythophthora.

8. The method of claim 7, wherein said Phythophthora is Phytophthora infestans and the primer set comprises oligonucleotides having the sequences 5'-CGATTCAAATGCCAAGCTAAAG-3' (SEQ ID NO: 1) and 5'-GCATCGATGAAGAACGCAGC-3' (SEQ ID NO: 2).

9. The method of claim 7, wherein said Phythophthora is Phytophthora erythroseptica and the primer set comprises oligonucleotides having the sequences 5'-CTGTTCCGGCGTAAGCTGG-3' (SEQ ID NO: 3) and 5'-TCCTCCGCTTATTGATATGC-3' (SEQ ID NO: 4).

10. The method of claim 7, wherein said Phythophthora is Phytophthora nicotianae and the primer set comprises oligonucleotides having the sequences 5'-ATTCAAAAGCCAAGCCACCG-3' (SEQ ID NO: 5) and 5'-GCATCGATGAAGAACGCAGC-3' (SEQ ID NO: 2).

Referenced Cited
Foreign Patent Documents
9529260 November 1995 WOX
Other references
  • Ersek et al., "PCR Amplification of Species-Specific DNA Sequences Can Distinguish Among Phytophthora Species", Applied and Environmental Microbiology, vol. 60(7), pp. 2616-2621, Jul. 1994. Niepold et al., "Application of the PCR Technique to Detect Phytophthora infestans in Potato Tubers and Leaves", Microbiol. Res., vol. 150, pp. 379-385, 1995. (Only Abstract). Niepold et al. Application of the PCR technique to detect phytophthora infestans in potato tubers and leaves. Microbiological Research vol. 150(4), pp. 379-385. (Only Abstract), 1995. O'Donnell Ribosomal DNA internal transcribed spacers are highly divergent in the phytopathogenic ascomycete Fusarium sambucinum. Curr. Genet. vol. 22, pp. 213-220, 1992. Wildman et al. Studies on the secondary structure of 5.8S rRNA from a Thermophile, Thermomyces Ianuginosus. J. Biol. Chemistry. vol. 256, pp. 5675-5682, 1981. Tooley et al. Development of PCR primers from internal transcribed spacer region 2 for detection of Phytophthora species infecting potatos. Applied and Environmental Microbiology, vol. 63(4), pp. 1467-1475, 1997. Database search Accession NO: Y08674 and Y08670, 1997.
Patent History
Patent number: 5874221
Type: Grant
Filed: Aug 28, 1996
Date of Patent: Feb 23, 1999
Assignee: The United States of America as represented by the Secretary of Agriculture (Washington, DC)
Inventors: Paul Tooley (Frederick, MD), Britt Bunyard (Frederick, MD), Marie Carras (Myersville, MD), Efstathios Hatziloukas (Frederick, MD)
Primary Examiner: Kenneth R. Horlick
Assistant Examiner: Joyce Tung
Attorneys: M. Howard Silverstein, John Fado, Janelle S. Graeter
Application Number: 8/704,207
Classifications
Current U.S. Class: 435/6; Absidia (435/912); 536/221; 536/243; 536/253; 536/2433
International Classification: C12P 1934; C07H 2104; C07H 2100;